The characteristic of immune parameters in zhikong scallop Chlamys farreri and bay scallop Argopecten irradians ISJ 11: 47-53, 2014 ISSN 1824-307X RESEARCH REPORT Biochemical biomarkers of Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) in exposure to TiO2 nanoparticles N Memarizadeh1, M Ghadamyari1, M Adeli2,3, K Talebi4 1Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran 2Department of Chemistry, Faculty of Science, University of Lorestan, Khoramabad, Iran 3Department of Chemistry, Sharif University of Technology, Tehran, Iran 4Department of Plant Protection, Faculty of Agriculture, University of Tehran, Karaj, Iran Accepted January 28, 2014 Abstract Biochemical biomarkers and bioassays, due to their assumed immediate response after acute exposure of the organism to the stressor, are useful tools to gauging anthropogenic impacts. The toxicity of TiO2-nanoparticles (TiO2-NPs) on the Glyphodes pyloalis Walker was assessed and the LC50 value obtained as 660.85 mg/L. The in vivo responses of G. pyloalis to sub-lethal concentrations of TiO2-NPs were surveyed by monitoring the activity of general esterases (EST), peroxidase (POD) and glutathione S-transferase (GST), as biochemical biomarkers. Activity of these biomarkers affected by exposure to TiO2-NPs and this could lead to the mortality or sub-lethal impacts. The effect of TiO2-NPs concentrations on the activity of these enzymes was correlated to the exposure time. The activity of EST and GST was significantly decreased compared to the control, after 24 h of treatments. By increasing exposure time, the expression of EST and GST was significantly increased. More POD expression was occurred at low concentrations (i.e. LC20 and LC30); however, at high concentrations, less POD activity obtained. It can be concluded that these enzymes are good early indicator of toxicity and in conjunction with acute toxicity studies allow adverse effects of TiO2-NPs to be predicted and managed. Key Words: TiO2-NPs; biomarker; Glyphodes pyloalis; esterases; glutathione S-transferase; peroxidase Introduction Nanoparticles (NPs) are particles with a diameter between 1 and 100 nm (Buzea et al., 2007). They show unique physico-chemical properties which differ from those of their respective bulk materials; such as large surface area, charge and shape (Handy et al., 2008). NPs can be utilized in the agricultural practices and this can be result in both risks and benefits for the ecosystem. Therefore, the evaluation of the safety of NPs in the environment looks very important (Kahru et al., 2008; Cattaneo et al., 2009). Due to unique characteristics of manufactured NPs of titanium dioxide (TiO2); such as chemically and biologically inert, stable toward corrosion and photocatalytic property, they are widely produced and consumed. Hence, the potential uses of TiO2-NPs in various fields subjected them to the ___________________________________________________________________________ Corresponding author Mohammad Ghadamyari Department of Plant Protection Faculty of Agricultural Sciences University of Guilan, Rasht, Iran E-mail: ghadamyari@guilan.ac.ir ecotoxicological studies (Linhua et al., 2009). Cytotoxicity, phytotoxicity, lung inflammation and oxidative stress in mammals, plants and microorganisms have been reported as side effects of TiO2-NPs (Ferin et al., 1992; Warheit et al., 2007; Wang et al. 2007, 2009; Clemente et al., 2012). Despite the fact that TiO2 has been classified as innocuous to the organisms (WHO, 1996); recently the International Agency for Research on Cancer (IARC) has classified this material as “possibly carcinogenic to humans” (IARC, 2010). Till now, the vast majority of nanoecotoxicological studies with TiO2-NPs have been focused on their toxicity to aquatic organisms (Clemente et al., 2012). Lovern and Klaper (2006) reported that Daphnia magna exposed to filtered TiO2-NPs showed 100 % mortality at 10 mg/L; whereas, sonicated TiO2-NPs caused only 9 % mortality at 500 mg/L. Federici et al. (2007) concluded that TiO2-NPs caused sub-lethal toxicity in rainbow trout (Oncorhynchus mykiss) involved oxidative stress, organ pathologies, and the induction of antioxidant defense system such as reduced glutathione (GSH). Zhu et al. (2008) - 47 compared the toxicity of several metal oxide NPs with their bulk counterparts to early developmental stages of zebrafish (Danio rerio) and showed that neither TiO2-NPs nor bulk TiO2 caused any toxicity to zebrafish embryos and larvae. Klaper et al. (2009) demonstrated that GST and catalase (CAT) are appropriate early biomarkers for prediction of physiological impacts and future toxicity of NPs to Daphnia. Linhua et al. (2009) showed that superoxide dismutase (SOD), CAT and POD activities and lipid peroxidation (LPO) levels in various tissues of carps varied with the concentration and exposure time. Jianhui et al. (2005) formulated dimethomorph with sodium dodecyl sulfate (SDS)/TiO2/Ag nanomaterial as a photodegradable nanofungicide. Guan et al. (2008) also used the same nanomaterials to formulation of imidacloprid. Furthermore, they produced W/TiO2/avermectin photodegradable microcapsules (Guan et al., 2010). Despite the investigation on nanoecotoxicological studies with aquatic organisms; so far, no published reports are available on acute toxicity and biochemical alteration (i.e., enzyme activities) caused by exposure of insect to TiO2-NPs. Thus, due to potential for application of TiO2-NPs to formulation of photodegradable pesticides; we aimed to make the ecotoxicological assessment of TiO2-NPs exposure to an insect pest model species, Glyphodes pyloalis. ESTs are detoxification enzymes which are involved in the insect physiology, metabolism, and xenobiotic detoxification (Ishaaya, 1993). Glutathione S-transferases (GSTs) are multifunctional enzymes in the phase II of pesticides metabolism (Ezemonye and Tongo, 2010). POD is an antioxidant enzyme which catalyzes the oxidation by hydrogen peroxide of a number of xenobiotics (Linhua et al., 2009). Because of the lack of knowledge about responses of insect EST, GST and POD to TiO2-NPs, these enzyme activities were evaluated in G. pyloalis treated with TiO2-NPs at different exposure times to predict the impact of these NPs at sub-lethal levels. Materials and methods Chemicals Glycerol, ethanol, triton X-100, H2O2, bovine serum albumin, α-naphthyl acetate (α-NA), β-naphthyl acetate (β-NA), Reduced Glutathione (GSH), 1-choloro-2,4-dinitrobenzene (CDNB), 1,2-Dichloro-4-nitrobenzene (DCNB), Bromophenol blue, Ethylene diamine tetraacetic acid (EDTA), Tris and acetic acid were purchased from Merck (Germany). Titanium isopropoxide (Ti(i-Pro)4) and guaiacol were purchased from Sigma-Aldrich (St Louis, Missouri, USA). Fast blue RR salt was bought from Fluka (Buchs, Switzerland). Insects The Glyphodes pyloalis was collected from infested mulberry orchards in the vicinity of Rasht, Iran. Mass rearing of insects was carried out in the laboratory, under controlled conditions with 25 ± 2 °C, 70 ± 10 % RH, and 16:8 L:D. Newly-ecdysed fifth instar larvae of G. pyloalis were used for bioassay experiments. Synthesis of TiO2-NPs TiO2-NPs were prepared according to the method of Trung et al. (2003) by hydrolyzing titanium isopropoxide which was added drop by drop into stock solution (i.e. ethanol and acetic acid in a ratio of 8:3 v/v with glycerol) at 10 °C, followed by rigorous stirring under an argon atmosphere for 3 h. Then, the solutions were heated at 60 °C for 5 h or until the gelling reaction was completed. The dried precipitates were heated at 400 °C for 10 h, at the heating rate of 1 °C/min. Bioassay with TiO2-NPs suspension The toxicity of TiO2-NPs suspension in water was assayed to newly-ecdysed fifth instar larvae of G. pyloalis using the leaf dip bioassay (Memarizadeh et al., 2011). Five serial dilutions of TiO2-NPs suspensions (250, 500, 800, 1000 and 1200 mg/L) were prepared and then sonicated for 30 min in a bath type sonicator (100 W, 40 kHz) to disperse the nanoparticles. Mulberry leaf discs (diameter 3.5 cm) were immersed in the dilutions for 45s. After drying of leaf discs, petioles of them were placed in a vial containing water to provide moisture. Up to 5 synchronized fifth instar larvae of G. pyloalis were placed on each treated leaf disk. Mortality was assessed after the treated larvae were maintained at 25 ± 2 °C, 70 ± 10 R.H. for 48 h. Each experiment was replicated ten times. The criterion for death was that a larva did not move when prodded with a camel’s hair brush. Treatment The newly-ecdysed fifth instar larvae of G. pyloalis were treated by different concentrations of TiO2-NPs suspensions (0, 290, 380, 475, 565 and 665 mg/L, equivalent to control, LC10, LC20, LC30, LC40 and LC50, respectively). Treatments were conducted according to the bioassay method. Over 3 days after treatments, representative samples were taken from survived larvae. The collected samples were placed in a deep freezer at -20 °C until biochemical assays were performed. Preparation of samples For determining EST activity, one larva was homogenized in 150 µl of 0.1 M phosphate buffer, pH 7.0 containing 0.05 % (v/v) Triton X-100 using a glass hand-held homogenizer on ice. After homogenization, they were centrifuged at 12,000×g for 15 min at 4 °C and resulted supernatant was used in the assay. For GST assay, enzyme preparation was similar to that previously mentioned for EST; however without Triton X-100. For POD assay, each larva was homogenized in 150 μl of 50 mM phosphate buffer (pH 7.4) containing 0.1 mM EDTA. Once homogenized, they were centrifuged at 12,000×g for 15 min at 4 °C and resulted supernatant was used in POD assay. Determining POD activity The 1 mL reaction mixture was consisted of 450 µl of 50 mM phosphate buffer (pH 7.4) containing 45 mM guaiacol, and 100 µl of prepared supernatant. Adding 450 µl of 50 mM phosphate buffer (pH 7.4) 48 Table 1 Log dose probit-mortality data for TiO2-NPs against G. pyloalis after 48 h n LC10(95% CI)a LC20 (95% CI)a LC30 (95% CI)a LC40 (95% CI)a LC50 (95% CI)a Slope ±SE χ 2 (df)b G. pyloalis 250 290.86 (151.02- 394.95) 385.51 (237.99- 492.06) 472.34 (326.8- 582.86) 561.88 (422.32- 683.51) 660.85 (525.57- 810.02) 3.59± 0.42 6.25 (3) aThe LC values are expressed as part per million (ppm) and their 95 % confidence intervals (95 % CI) bThe value of p > χ2 larger than or equal to 0.05 indicates a significant fit between the observed and expected regression lines containing 225 mM H2O2, the reaction was initiated. Then, the oxidation of guaiacol was followed at 470 nm with a spectrophotometer (Cary 3) using an extinction coefficient of 26.6 mM-1cm-1 (Bergmeyer, 1974). The POD activity was expressed as μmol.min-1mg protein-1. Determining EST activity Esterase activity was determined based on the van Asperen (1962) method. α-NA and β-NA were used as substrates. 12 µl of supernatant were added to per well of a microplate which containing 113 µl phosphate buffer (pH 7.0). After 3 min, adding 50 µl of 1.8 mM substrate solution, the reaction was initiated. Following the addition of 50 µl of the fast blue RR salt, absorbance at 450 and 540 nm were measured in a microplate reader (Awareness Technology Inc., Florida, USA) for α-NA and β-NA, respectively. The formation of the α-naphthol- and β-naphthol-fast blue RR dye complex was converted to a specific activity using standard curves, which were obtained from different concentrations of α-naphthol and β-naphthol mixed with fast blue RR salt (0.075 %), respectively (Miller and Karn, 1980). The EST activity was expressed as nmol.min-1mg protein-1. Determining GST activity GST assays were conducted according to the method of Habig et al. (1974), using CDNB and DCNB as substrates. 15 µl supernatant, 100 µl of 1.2 mM substrate solution and 100 µl of 10 mM GSH were added to per well of a microplate. Enzyme activity was determined by continuously monitoring the change in absorbance at 340 nm for 5 min at 25 °C with a microplate reader (Awareness Technology Inc.). The GST activity was expressed as μmol.min-1mg protein-1. Determining protein concentration Protein concentrations were estimated by the Bradford (1976) method, using bovine serum albumin as standard. Statistical analysis Three replicates were conducted for all the biochemical assays and data were subjected to analysis of variance (ANOVA). Statistical analyses were performed at p = 0.05 by Tukey’s test using the SAS software. Results Bioassay results Table 1 shows the median lethal concentration (LC50), sub-lethal endpoints and the 95 % confidence limits which calculated from probit regression using the POLO-PC computer program (LeOra, 1987) and based on Finney (1971). POD activity The level of POD differed significantly among treatments (Fig. 1). 24 h after G. pyloalis larvae treatment with LC20 and LC30 concentrations of TiO2-NPs, the highest POD levels were observed. Results showed that by increasing the sub-lethal concentrations from LC10 to LC30 and by increasing exposure time, POD activity was significantly increased compared to the control. LC50 concentration of TiO2-NPs significantly decreased POD activities. However, they were not significantly changed at LC40 concentration in comparison to the control group (Fig. 1). Fig. 1 Peroxidase (POD) activity in G. pyloalis exposed to sub-lethal concentrations of TiO2-NPs at different time intervals. Different letters indicate that the specific activity of enzymes is significantly different from each other by Tukey’s test (p < 0.05). EST activity EST activities of G. pyloalis treated with sub- lethal concentrations of TiO2-NPs are presented in 49 Figure 2. Analysis of variance showed that esterase activity, using both α-NA and β-NA, significantly affected by: 1) the TiO2-NPs concentrations, 2) time of exposure to TiO2-NPs and 3) interplay effect of concentration and exposure time. This means that the effect of TiO2-NPs concentrations on the esterase activity is correlated to the length of exposure time. Assessment of EST activity in treatments after 24 h showed that TiO2-NPs led to inhibition of these enzymes and thus decreased their activities. However, when α-NA was used as substrate, by increasing the time of exposure to LC10, LC20 and LC30 concentrations, the EST activities significantly increased (Fig. 2). A B Fig. 2 Comparison of esterase activity in G. pyloalis exposure to different concentrations of TiO2-NPs over 3 days, using α-NA (A) and β-NA (B) as substrates. Means followed by similar letters showed no significantly difference from each other by Tukey’s test (p < 0.05). GST activity GST activities of G. pyloalis treated by sub- lethal concentrations of TiO2-NPs are presented in Figure 3. When CDNB was used as substrate, increasing TiO2-NPs concentrations led to significant reduction in GST activities after 24 h; however, this reduction was steeper than when DCNB was used in the assay. Furthermore, as depicted in Figure 3, by increasing the exposure time of G. pyloalis to LC20, LC30 and LC40 of TiO2-NPs, GST activity significantly increased. Results of analysis of variance showed that using both CDNB and DCNB, GST activity significantly affected by: 1) the TiO2- NPs concentrations, 2) exposure time of larvae to TiO2-NPs and 3) interplay effect of concentrations and exposure time. This means that the increasing TiO2-NPs concentrations over exposure time could be enhance the conjugation of GSH to these NPs and thus the enzyme activity was affected. A B Fig. 3 Comparison of GST activity in G. pyloalis exposure to different concentrations of TiO2-NPs over 3 days, using CDNB (A) and DCNB (B) as substrates. Means followed by similar letters showed no significantly difference from each other by Tukey’s test (p < 0.05). Discussion Since the growth in use of NPs and nanotechnology in various fields mainly in agriculture is inevitable, ongoing study of nanotoxicity will be necessary in order to avoid their unpredictable complications (Clemente et al., 2012). To minimize the adverse environmental effects of manufactured and engineered nanomaterials, it is critical to have a good understanding of their toxic potential (Handy et al., 2008). The dose-mortality data for TiO2-NPs generated in the present study showed a LC50 value of 660.85 mg TiO2-NPs suspension liter −1 in G. pyloalis larvae. Following the bioassay results, the G. pyloalis larvae were exposed to 290, 380, 475, 565 and 665 mg/L TiO2-NPs as LC10, LC20, LC30, LC40 and LC50, respectively. The experiments were designed to allow sub-lethal physiological effects of TiO2-NPs. The exposure time of 3 days was chosen to enable some biochemical responses of treated insects. Lovern and Kapler (2006) reported an LC50 of 5.5 ppm in D. magna which exposed to filtered TiO2- NPs for 48 h. Kim et al. (2010) reported a 70 % mortality rate in D. magna exposed to 5 mg/L of Sigma Aldrich TiO2-NPs for 21 days. Wang et al. (2009) were estimated 80 mg/L of TiO2-NPs as LC50 50 and showed that TiO2-NPs are more toxic than their bulk counterparts to the Caenorhabditis elegans. Acute toxicity assays will not be able to provide sufficient information on the interaction of nanomaterials and organisms. Whereas, sub-lethal effects can be indicated the impacts of NPs on the physiology and survival of organisms. Biochemical biomarkers as indicators of sub-lethal effects of a stressor can be used to early warning of population level impacts (De Coen and Janssen, 1997; klaper et al., 2009). Biomarkers indicative of neurotoxicity (EST), oxidative stress (POD) and phase II biotransformation of xenobiotics (GST), as well as general mortality have been linked to population- level endpoints (Jemec et al., 2007; Paskerova et al., 2012). Usage of these biomarkers in risk assessments is suitable diagnostic tool for the detection of specific contaminants well before real adverse effects can occur (Nascimento et al., 2008). Pollutants may increase the intracellular formation of reactive oxygen species (ROS) which have been reported to affect the physiology, growth, and survival of organisms (Filho, 1996; Pandey et al., 2003). POD is the key enzyme in antioxidant defense systems to convert the resulting free radicals H2O2 to water and oxygen (Linhua et al., 2009). Over 3 time points of treatments, by increasing the concentration of TiO2-NPs from LC10 to LC30, the POD activities were increased and then at LC and LC concentrations were decreased. 40 50 Therefore, results of the present study demonstrate that enhanced activities of POD at low concentrations could lead to the elimination of ROS. This also could be an indication that exposure to low concentrations would yield less mortality in these treatments. Because of the importance of general ESTs in the insect physiology, metabolism and detoxification of xenobiotics, in this study the possible role of these enzymes as a biomarker to determination of NPs toxicity was investigated (Ishaaya, 1993; Memarizadeh et al., 2013). Results of the present work showed that by increasing TiO2-NPs concentration ESTs activities were decreased due to their inhibition. Also the LC50 concentration of TiO2- NPs had the highest inhibition on the EST activity, when α-NA was used as substrate. Since, an effective inhibition of α-esterases occurs at LC50 concentration; these enzymes may be as a good indicator of the TiO2-NPs toxicity. GSTs are multifunctional enzymes of the phase II biotransformation system which play a key role in metabolism of a broad variety of xenobiotics and endogenous compounds (Ezemonye and Tongo, 2010; Ezeji et al., 2012; Memarizadeh et al., 2013; Zamani et al., 2014). GSTs are able to conjugate the xenobiotics and their metabolites to the tripeptide glutathione (GSH) and making them soluble for easy excretion (Oakley, 2011). In addition to detoxification role of these enzymes, GSH also has antioxidant properties. Thus, GSH as a general stress indicator is a more useful diagnostic tool. Our study revealed that the GST activity significantly affected by exposure to TiO2-NPs. So, increased TiO2-NPs concentrations led to significant reduction in GST activities. Results also showed that the LC50 concentration of TiO2-NPs had the highest impact on the GST activity. Data demonstrated that activities of detoxification and antioxidant enzymes altered by exposure G. pyloalis to TiO2-NPs and this can be lead to the mortality or sub-lethal impacts. Furthermore, GST, EST and POD activities mainly affected by LC50 concentration and thus, this concentration is a good early indicator of TiO2-NPs toxicity. The sub-acute toxicity of TiO2-NPs to carp (Cyprinus carpio) was assessed by Linhua et al. (2009). They showed that 100 and 200 mg/L TiO2- NPs caused the significant decrease in SOD, CAT and POD activities suggesting that the fish exposed to TiO2-NPs suffered from the oxidative stress. As each biomarker may have different sensitivities depending on the mode of action of the chemical (Jemec et al., 2007); the use of multiple biomarkers to evaluate toxicity of NPs for insects will provide the most suitable tool to indicate the potential impacts of them. Conclusion The results of this study suggest that sub-lethal effects of TiO2-NPs to G. pyloalis are related to concentration and length of exposure time. EST, GST and POD as biochemical biomarkers are useful diagnostic tools to determination of NPs toxicity. POD activity in different treatments indicates that these particles are causing oxidative stress; especially at a lower concentration than the LC50 concentration. 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